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SYBORG Report Summary

Project ID: 637675
Funded under: H2020-EU.1.1.

Periodic Reporting for period 1 - SYBORG (combining SYnthetic Biology and chemistry to create novel CO2-fixing enzymes, ORGanelles and ORGanisms)

Reporting period: 2015-05-01 to 2016-10-31

Summary of the context and overall objectives of the project

Atmospheric carbon dioxide (CO2) is a potent greenhouse gas, but also considered as an important carbon feedstock of the future. The sustainable capture and utilization of CO2 will be crucial to realize a green economy that is independent from petrochemical sources. Yet, in respect to anthropogenic CO2 emissions, our technical capabilities to recapture atmospheric CO2 are lacking behind. Chemistry still struggles with developing catalysts and processes that allow the conversion of atmospheric CO2 under mild conditions into a multi-carbon, value-added product in a carbon-neutral, or carbon-positive, sustainable way.

The project "SYBORG" explores novel ways to activate and bind atmospheric CO2 with biochemical and synthetic biological approaches. To that end, the project focuses on reductive carboxylation of enoyl-CoA esters, a completely novel principle of enzymatic CO2-fixation that was discovered only recently. This new principle is one the most efficient CO2-fixation reactions described in biology so far. The specific objectives of the project "SYBORG" are: 1) Understanding and engineering the mechanism of reductive carboxylation for new CO2-fixation reactions, 2) Designing and realizing synthetic pathways for CO2-fixation based on reductive carboxylation, and 3) Implementing synthetic CO2-fixation pathways based on reductive carboxylation into selected model organisms.

In summary, with project "SYBORG" we will break new grounds in understanding and engineering biological systems and mechanisms for an efficient CO2-fixation to convert the waste product CO2 into useful compounds. The long-term goal is to develop new ways towards artificial photosynthesis.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

OBJECTIVE 1: Understanding and engineering the mechanism of reductive carboxylation.
We established methods to synthesize a diverse library of potential substrates to screen and engineer novel CO2-fixation reactions in the family of enoyl-CoA reductases/carboxylases (Peter et al. Molecules 2016). We used this library of substrates to assess the substrate specificity in enoyl-CoA carboxylases/reductases with a newly developed mass spectrometry based screen. Bioinformatics and site directed mutagenesis allowed us to identify three amino acid residues that determine substrate specificity in this class of CO2-fixing enzymes. This allowed us to engineer and extend the substrate spectrum of reductive carboxylases to create new CO2-fixation reactions that can provide new building blocks in polyketide biosynthesis (Vögeli, unpublished) as well as for the use in synthetic CO2-fixation pathways (Schwander et al. Science 2016).

OBJECTIVE 2: Designing and realizing synthetic pathways for CO2-fixation based on reductive carboxylation.
We realized an artificial pathway for CO2-fixation, the CETCH cycle. The CETCH cycle was drafted by ‘metabolic retrosynthesis’, established with seventeen enzymes originating from nine different organisms of all three domains of life, and optimized in several rounds by enzyme engineering and metabolic proofreading. The CETCH cycle fixes CO2 in vitro at a rate of 5 nmol min-1 mg-1 protein (Schwander et al. Science 2016). We demon-strated that complex metabolic networks can be designed and realized by the principle of ‘metabolic retrosynthesis’ following basic (bio)chemical considerations, but that the operation of these synthetic networks needs further optimization by including biological design principles. Following these rules, we were able to realize a completely synthetic pathway for CO2-fixation that shows favorable thermodynamics compared to naturally evolved CO2-fixation pathways (requiring less ATP per CO2 fixed than photosynthetic CO2-fixation of plants).

OBJECTIVE 3: Implementing synthetic CO2-fixation pathways based on reductive carboxylation into seleceted model organisms.
We started to establish microbial platforms to establish the CETCH cycle and other synthetic CO2-fixation pathways into Methylobacterium extorquens.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

We succeeded for the first time to dissect individual catalytic events steps of reductive carboxylases, the most efficient CO2-converting biocatalyst known so far. This detailed understanding of an efficient biological CO2-fixation reaction will allow us to derive the (bio)chemical principles of CO2 binding and activation for the development of novel catalytic strategies in chemistry. On a more applied biotechnological aspect, we could already manipulate these enzymes for the production of new building blocks for polyketide-antibiotics from CO2.

We have used reductive carboxylases to build a completely novel, artificial pathway for CO2-fixation in vitro that is 20% more efficient than the CO2-fixation mechanisms used in natural photosynthesis. This shows that it is in principle possible to design highly efficient, artificial photosynthetic processes in a rational fashion. We expect that the CETCH cycle (and other not yet realized CO2-fixation cycles) will open further research directions in bottom-up and synthetic biology. The realization of improved CO2-fixation cycles has broader implications in respect to developing novel strategies in capturing and converting the climate gas CO2, increasing photosynthetic yield and crop productivity, but also in respect to the acceptance of synthetic biology and genetic modified organisms.
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